Protein-protein interactions Courtesy of Sarah Teichmann & Jose B. Pereira-Leal MRC Laboratory of Molecular Biology, Cambridge, UK EMBL-EBI

Stable complex Transient Interaction Transient Signaling Complex Rap1A – cRaf1 Interface 1310 Å 2 Stable complex: homodimeric citrate synthase Interface 4890 Å 2 Hydrophobic interfaces “Hydrophilic” interfaces Stable vs. transient protein-protein interactions Multi-domain protein

Stable vs. transient protein-protein interactions

Interface Constraints in Multi-domain Proteins 42% 43% 45% No. proteins in S. cerevisiae: Average identity S. cerevisiae- S. pombe:

Summary Sequence conservation: Stable complexes > transient > other Co-expression/co-regulation: Stable complexes > transient

Using publicly available interaction data 1.There are interactors for your protein in the literature 2.There are databases of interactions where your protein may appear 3.There are homologues of your protein in the protein interaction databases 4.You can predict interactors by other means? 5.This failing, at this point you go back to the bench… Are there know interaction partners for you pet protein? Check if:

Using publicly available interaction data Problems: Low coverage Does not include results from high throughput experiments Gene names may not be consistent 1.Are there interactors for my protein in the literature ?

Using publicly available interaction data 2. Are there databases of interactions where my protein may appear? Some DBs: BIND, MINT (General) + organism specific databases (e.g. MIPS/CYGD) Caution! Check: -the experimental methods used to identify the interaction (e.g. high error rate in large scale yeast-two hybrids) -check the method used to incorporate the interaction in the database (e.g. manual curation vs. literature mining using “intelligent” algorithms)

Using publicly available interaction data 3. Are there homologues of my protein in the protein interaction databases? We are assuming that protein interactions are conserved in evolution Plenty of evidence that they are… BUT, how do you define homologous/orthologous ? Make sure that you understand the limits of such “prediction”: two single-gene family products interact in one organisms, and they also exist as single gene-family products in another genome --> potentially good prediction -but the original interactions was identified in a large scale Y2H, is not supported by any other observation and one of the proteins has 133 described interactors in that experiment… --> likely a false positive (you learned nothing about your protein!)

Computational Prediction of protein interactions (functional associations) Caution: Computational methods are good at finding functional associations A functional association is not the same thing as a physical interaction Since : we don’t know how many of the experimentally derived interactions are true/biologically significant We don’t know how many interactions exist Impossible to determine how good predictions REALLY are (this becomes more important as the number of predictions you make increase [automation]) Apparently no one in the world ever bothered to look at your favorite protein… now what?

Experimental techniques Yeast two-hybrid screens MS analysis of tagged complexes Correlated mRNA expression levels Synthetic lethality

Experimental techniques Yeast two-hybrid screens MS analysis of tagged complexes Correlated mRNA expression levels Synthetic lethality Microarray timecourse of 1 ribosomal protein mRNA expression level (ratio) Time-> Random relationship from 18M Close relationship from 18M (2 Interacting Ribosomal Proteins) Predict Functional Interaction of Unknown Member of Cluster

Experimental techniques Yeast two-hybrid screens MS analysis of tagged complexes Correlated mRNA expression levels Synthetic lethality

How good is the data? (von Mering et al., Nature 417:399)

”We estimate that more than half of all current high-throughput interaction data are spurious”

Computational prediction of protein interactions Tryptophan synthetase   fusion 1PII TrpCTrpF Fused in E.coli Unfused in some other genomes (Synechocystis sp. and Thermotoga maritima.) Enright et al (1999) Nature 409:86 Marcotte et al (1999) Science 285: 751 Gene fusion events

Pellegrini et al (1999) PNAS 96: 4285 Computational prediction of protein interactions Phylogenetic profiles

Computational prediction of protein interactions Conservation of Gene neighborhood e.g. operons in bacteria Not really applicable to eukaryotes, except, to some extent, C. elegans However, there is hope for eukaryotes: -adjacent genes are frequently co-expressed (co-regulated) -co-regulated proteins are likely to be functionally associated  maybe this principle may be used for prediction of interactions

Computational prediction of protein interactions Mirror trees Proteins that physically interact tend to co-evolve Pazos and Valencia (2001) Protein eng. 14: 609

Computational prediction of protein interactions Pre-computed predictions: where to find them?

The Big Picture

Scale-free networks (Ravasz et al., Science 297:1551)

Some examples of systems with a scale invariant organization Broccoli World-wide web (some) Food webs Social networks Roads

Scale free behavior in protein interaction networks: scale free or scale invariance  self-similarity Scale invariance gives insight into robustness of biological systems

Modular networks

Hierarchical networks

Identification of functional modules from protein interaction data Graph theory formalisms Custering Messy data Functional modules Pereiral-Leal, Enright and Ouzounis (2003) Proteins in press

From functional modules to pathways Canonical pathways Pereiral-Leal, Enright and Ouzounis (2003) Proteins in press

Prediction of the molecular basis of protein interactions So.. You know your two proteins interact… do you want to know how?

Molecular basis of protein interaction “Tree determinant residues” Rab Ras Rho Arf Ran x REP + _ MSA Prediction Experimental tests Pereira-Leal and Seabra (2001) J. Mol. Biol. Pereira-Leal et al (2003) Biochem. Biophys. Res. Com.

Molecular basis of protein interaction “Tree determinant residues” Continued… Sequence Space algorithm Casari et al (1995) Nat. Struct. Biol 2(2) AMAS (part of a bigger package)

Molecular basis of protein interaction In silico docking Requires 3D structures of components Conformational changes cannot be considered (rigid body)